303 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 supplemented with 1.25 µM Cal-520 (Abcam) and 0.02% Pluronic F-127 solution (Sigma Aldrich). Spontaneous Ca2+ transients were recorded three weeks after plating. Video streams of Cal-520 (green channel) lasting 10 seconds at 33 fps were automatically scanned by a Leica Thunder microscope. Image analysis was conducted using Cyteseer (Vala Sciences, California, USA), as previously described.30,31 The physiological parameters; decay time, peak value (normalized area under the peak trace), rise time, and Ca2+ Transient Duration (CTD) on 10% (CTD10) and 90% (CTD90) of the peak width and beats per minute were automatically calculated for each time series. Spheroid immunolabeling and confocal imaging. For immunofluorescent stainings, whole 3D microtissues were used to image at a single-cell resolution, adapted from a recently published protocol.32 In short, 1 mil. hiPSC-CMs were seeded in 6 wells (ultra-low attachment, corning). After 3 weeks of culturing, half of the medium was carefully aspirated and 1 mL of cold phosphate-buffered saline (PBS) was added. The hCSs were carefully collected by BSA-coated tips (Roche) into BSA-coated tubes. The hCSs were washed with 10 mL ice-cold PBS and spun down for 3 min at 70xg and 4° C. Next, the hCSs were resuspended in 1 mL 4% paraformaldehyde (PFA, Santa Cruz) solution and fixed for 45 min at 4°C. After the fixation, the hCSs were spun down for 3 min at 70xg and 4° C, and washed with 10 mL ice-cold PBS 3 times. HCSs were then blocked for 15 min at 4°C in spheroid washing buffer (SWB), consisting of 0,1% Triton X-100 (Sigma), 0,2% of 10% (w/v) SDS (Sigma), and 0,2% bovine serum albumin (BSA, Sigma) in PBS (stored at 4°C up to 2 weeks). After fixation and blocking, the hCSs were transferred to a 24-well suspension plate where immunolabeling was performed. hCSs were incubated with various primary antibodies ( Supplementary Table 1). DNA Hoechst staining was used for nuclei labelling (Thermo Fisher Scientific) and was added together with the secondary antibodies (0.5 µg/mL Goat Anti-mouse 488, and 0.5 µg/mL goat anti-rabbit-568, Thermo Fisher Scientific). For the direct Nile Red labelling, 10 μg/ml Nile Red (Sigma) was added to the secondary antibody mix. Specimens were mounted on microscope glasses using FUnGI consisting of 50% (v/v) glycerol (Sigma), 9.4% (v/v) dH2O, 10.6 mM tris base (Roche), 1.1 mM EDTA (Sigma), 2.5 M fructose (Sigma) and 2.5 M urea (Sigma). Fluorescent imaging was done using an SP8 Confocal Microscope (Leica). Optical sectioning along the Z-axis was performed and the images collapsed into a single focal plane using the manufacturer’s software. Flow cytometry analysis. The hCSs were digested by TrypLE Select Enzyme for 30 min at 37°C. The single CMs were fixed with 4% paraformaldehyde (Santa Cruz) for 10 minutes at 37°C. Cells were then permeabilized using 1% ice-cold BSA-PBS and 0.3% Triton-X-100 for 30 minutes on ice. The primary antibody against alpha-actinin (0.25 µg/mL dilution)(mouse, A7811 MilliporeSigma) was incubated for 1 hour while the secondary antibody goat antimouse Alexa Fluor-488 (0.25 µg/mL dilution) (Thermo Fisher Scientific) was incubated with
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